Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-04 to 2018-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Deviations did not adversely affect the results of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

EC Number:

933-779-9

Molecular formula:

C10H16

IUPAC Name:

Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

Test material form:

liquid

Details on test material:

Name: [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene]
Public name: Ocimene PQ
EC numbers: 933-779-9 (pre-registration 222-081-3)
CAS numbers: n/a (pre-registration 3338-55-4)
Batch/Lot number: A170524D
Description: Colourless liquid
Expiry date: 06 June 2019
Purity: Considered as 100%

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Emerald Kalama Chemical Limited (Dans Road, WA8 0RF Widnes, Cheshire United Kingdom); Batch/Lot number: A170524D
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-07-02

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤70 RH%), under inert gas, protected from humidity (tight closed container)
- Stability under storage conditions: Stable
- Stability under test conditions: stable for 1 day when stored at room temperature, and at least 15 days when stored below -15°C.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid

OTHER SPECIFICS
- other information:
- Name of substance: Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene
- Public name: Ocimene PQ
- EC# 933-779-9 (pre-registration 222-081-3)
- CAS numbers: n/a (pre-registration 3338-55-4)
- Batch/Lot number: A170524D
- Expiry date: 06 June 2019
- Purity: Considered as 100%

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Wistar) rats

Details on species / strain selection:

The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. The Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were also used for the Dose Range Finding study (study code: 17/180-220PE).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany), from SPF colony)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult rats, approximately 11-13 weeks old at start of treatment
- Weight at study initiation: Males: 412–502 g, females: 242–317 g at onset of treatment
- Fasting period before study: Not specified
- Housing: Rodents were group-housed in Type II and III polycarbonate cages, up to 2 animals of the same sex and dose group/cage with the exception of the
mating and gestation/delivery period when they were paired or individually housed, respectively
- Diet (e.g. ad libitum): ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 840 33675 / 639 38520, Expiry date: 31 January 2019 / 30 April 2019) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand Gabriel Weg 16, D-59494 Soest, Germany) ad libitum
- Water (e.g. ad libitum): Tap water from the municipal supply, as for human consumption, was provided from a 500 mL bottle, ad libitum
- Acclimation period: At least 14 days

DETAILS OF FOOD AND WATER QUALITY:

Water quality control analysis was performed at least once every three months and microbiological assessment was performed monthly, by Veszprém
County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary). The diet supplier provided analytical certificates for the batches used. Food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0–25.0°C (target range 22 ± 3°C)
- Humidity (%): 26–69% (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2018-10-4 To: 2018-12-18

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is one of the possible routes of human exposure

Vehicle:

propylene glycol

Remarks:

propylene glycol with 1% polysorbate 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was formulated at appropriate concentrations (0, 12, 35, or 100 mg/mL for doses of 0, 60, 175, or 500 mg/Kg bw/day, respectively) in the vehicle (as a visibly stable homogenous formulation) and formulations were prepared daily. Dosing solutions were administered to the test material or vehicle-treated (control) animals at a constant volume of 5 mL/Kg bw (calculated and adjusted based on each animal’s most recent body weight).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on results of a short solubility test performed at the Test Facility, propylene glycol with 1% polysorbate 80 was selected as vehicle for this study in agreement with the Sponsor based on the formulation and analytical trials. The same vehicle was used in the Dose Range Finding (DRF) study.
- Concentration in vehicle: 0, 12, 35, or 100 mg/mL for doses of 0, 60, 175, or 500 mg/Kg bw/day, respectively.
- Amount of vehicle (if gavage): 5 mL/Kg
- Lot/batch no. (if required):

Name: Propylene glycol
Manufacturer: MERCK
Batch number: K49089078 / K50526178
Expiry date: 31 May 2022/31 August 2023
Storage conditions: Room temperature

Name (Synonym): Polysorbate 80 (Tween 80)
Manufacturer: Sigma-Aldrich
Batch number: BCBV5152
Expiry date: 30 June 2022
Storage conditions: Room temperature

- Purity: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test material formulations for concentration and homogeneity was performed using a validated HPLC-UV method (High Performance Liquid Chromatography with Ultraviolet detection). Duplicate samples were taken from the top, middle and bottom of the test item formulations three times during the study (during the first and last weeks and approximately midway during the treatment), one set to analyse and one set as a backup, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement. Formulation samples (both sets) were kept frozen (under -15 °C) until shipment. Samples were shipped on dry ice as soon as possible after collection for concentration and homogeneity measurement to the Principal Investigator.

Duration of treatment / exposure:

Males: 28 days (14 days pre-mating and 14 days mating/post-mating)
Females: 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13 days post-partum dosing).

Frequency of treatment:

Once daily (7 days/week)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 - Control

Doses / concentrations 2

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

Group 2 - Low Dose

Doses / concentrations 3

Dose / conc.:

175 mg/kg bw/day (nominal)

Remarks:

Group 3 - Mid Dose

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 4 - High Dose

No. of animals per sex per dose:

12/sex/dose (Two females were used as replacement on Day 3, one female on Day 4 and another female on Day 5. Two additional males were also included in the study to provide mating pairs for the replacement females on Day 4 and Day 5)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected by the study director in consultation with the sponsor based on available acute oral toxicity data (LD50 over 2000 mg/Kg bw in rats (Charles River Laboratories Hungary Kft., 2019; Study code: 17/180-001P) and results of the Dose Range Finding (DRF) study performed at the Test Facility (Charles River Laboratories Hungary Kft., 2019: Study code: 17/180-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. In the DRF study, test material related mortality was seen both at 1000 mg/Kg bw/day and at 600 mg/Kg bw/day. All four females dosed with 1000 mg/Kg bw/day were found dead, and in 600 mg/Kg bw dose group, one was female found dead, and the other females showed signs of systematic toxicity.

- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day 0). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.

- Fasting period before blood sampling for clinical biochemistry: overnight

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily (Not on days when detailed clinical observations were made), after treatment at approximately the same time with minor variations, or in the
afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. Animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult orprolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed at least weekly during the preexposure period, then on Day -1 for randomisation purposes, on Day 0 then at least weekly and at termination. Parental females were weighed on Gestation Days (GD) 0, 3, 7, 10, 14, 17 and 20 and on post-partum days (PPD) 0 (within 24 hours after parturition), 4, 7, 10, 13 and 14 (before termination).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Animal food consumption was determined by re-weighing the non-consumed diet weekly (on the days of body weight measurements).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (fasted (overnight period of food deprivation, after the litter had been culled))
- How many animals: 5 male and 5 female animals/group (Note: Due to the high mortality of the High dose females, only the 3 surviving females were sampled
and evaluated)
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy
- Animals fasted: Yes (fasted (overnight period of food deprivation, after the litter had been culled))
- How many animals: 5 male and 5 female animals/group (Note: Due to the high mortality of the High dose females, only the 3 surviving females were sampled
and evaluated)
- Parameters checked in table [No.3] were examined.

SERUM HORMONES: Yes
- Time of blood sample collection: PND14 (females) / PND13 (pups)
- Animals fasted: Yes
- How many animals: all surviving dams and at least two pups per litter on PND14 (females) / PND13 (pups). All adult males.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.4] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Last exposure week (males on Day 25; females on PPD9-12)
- Dose groups that were examined: 5 male and 5 female animals/group (Note: Due to the high mortality of the High dose females, only the 3 surviving females were sampled and evaluated)
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At termination, the surviving adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination. Gross necropsy was performed on all animals, irrespective of the date of death. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened, and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The pre-terminally euthanized and the found dead animals were examined similarly to the terminal animals.

At the time of termination, body weight and the weight of the following organs from all surviving adult animals were determined:

1) With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus

2) With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroid glands

Testes and epididymides were weighed paired. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported. The organ weights of the pre-terminally euthanized animal were measured similarly to the terminal animals. However, no organ weight measurement was performed in the animal found dead.

HISTOPATHOLOGY: Yes (see table 5)

The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, testes with epididymides in Bouin’s solution, and all other organs in 10% buffered formalin solution.

Thyroid glands from one male and one female PND 13 pup selected by the study director from each litter were preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage. For microscopic examination, the retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

For the adult animals, a detailed histological examination was performed as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group (Due to the high mortality of the High dose females, only the 3 surviving females were evaluated)),
• all found dead High Dose female animals,
• all organs where macroscopic findings (abnormalities) were seen,
• retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups and of all males that failed to sire and all females that failed to deliver healthy pups
• the kidney samples of all the remaining males,
• the stomach samples of remaining males and females.

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Other examinations:

Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs. The number of implantation sites and of corpora lutea were recorded in female animals as applicable. Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities, where possible.

Statistics:

For information on statistics please see the section 'Any other information on materials and methods incl. tables'.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were observed in any of the male animals during the study.

The following signs were observed in female rats:
In control group females, piloerection was present in 4 out of 10 animals. In the Low dose group, 6 out of 10 animals showed piloerection at some point during the study; from those 6 animals, 2 animals produced liquid faces (2 out of 10 evaluated animals), and among those two, in one animal hunched back was also observed (1 out of 10 evaluated animals). The observed changes were considered to be incidental or related to the dosing procedure, and not related to treatment.

Mid dose group females showed piloerection (8 out of 10 evaluated animals), hunched back (1 out of 10 evaluated animals), and noisy respiration (4 out of 10 evaluated animals). These changes were considered to be treatment-related, adverse changes.

Furthermore, brown discharge from the vulva was observed in one dam, and in another animal, thin fur on both forelimbs were also observed. These changes were considered to be incidental, and not treatment-related.

In the surviving High dose female animals, hunched back, piloerection, and noisy respiration was present (3 out of 3 animals), as well as red discharge from the noise in 1 out of 3 animals. These changes, due to their frequency and similarity to findings in the found dead animals, were considered to be treatment-related, adverse effects.

No clinical signs were observed in the two non-pregnant Control group females.

Piloerection was present for one occasion in 1 out of 2 animals with total intrauterine mortality in the Low dose group. Noisy respiration was present for one occasion in 1 out of 2 animals with total intrauterine mortality in the Mid dose group. These findings were considered to be incidental, and not treatment-related adverse effects.

In the non-littering High dose females (12 found dead animals and 1 animal with total intrauterine mortality), the symptoms of piloerection (11 out of 13 animals), noisy respiration (11 out of 13 animals), laboured respiration (4 out of 13 animals), hunched back (9 out of 13 animals), decreased activity (2 out of 13 animals), ataxia (1 out of 13 animals), recumbency (3 out of 13 animals), red discharge (5 out of 13 animals) and intermittent tremors (1 out of 13 animals) were present, and these findings were considered to be a treatment-related, adverse effects, although direct correlation with the death of the animal was not established for all of the animals found dead.

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment-related mortality was observed in the High dose (500 mg/Kg bw/day) females where 12 animals were found dead either at the beginning of the treatment (between Day 2-6, six animals in total: #4505, #4507, #4508, #4510, #4605 and #4607), or near the end of the pregnancies (Day 24, or between Day 35-38, six animals in total: #4502, #4506, #4509, #4512, #4608 and #4610).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
All body weight and body weight gain values were normal in all dose groups, with the exception of the period between Day 14 to Day 21, where, due to the significantly lower body weight gain data in the control group, the body weight gain data in the high dose group was found to be statistically significantly higher (p < 0.01), despite being normal for a weekly body weight gain. This finding was considered to be incidental and not related to treatment.

Females:
Body weight of the high dose group was normal during the pre-mating and mating period but lower body weight was observed during the gestation period (reaching statistical significance (p<0.05) on GD7) and during the lactation period. Body weight gain data for the same periods showed constantly lower body weight gain values for these animals, reaching statistical significance for the entire gestation period (where at least 9 high dose females were compared to the control females). While no statistically significant difference was observed during the lactation period, it should be noted that for these values, only the 3 surviving high dose females were compared to the control group. The decreased body weight and body weight gain in the high dose females was considered to be a treatment-related adverse effect. For the low- and mid-dose female animals, all body weight and body weight gain values were normal.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
Significantly lower (-9.1%, p< 0.01) food consumption was observed in the high dose animals at the beginning of the treatment (Day 0-7), which was considered to be a transient, treatment-related effect. After this period, there were no treatment-related differences in the mean daily food consumption in any treated groups when compared to the control animals. The measured values were within the normal range for this strain and age.

Females:
High dose females showed significantly lower food consumption values during the first two weeks of the treatment (-30.3%, p< 0.01), and during the gestation period (-19.8%, p< 0.01). For the 3 surviving animals, lower food consumption data was measured during the lactation period (-12.3%, not reaching statistical significance when calculated for the entire lactation period), and the difference between high dose and control on the second week was more pronounced (-16.8%). These reduced food consumption values were considered to be treatment-related effects. There were no treatent-related differences in the mean daily food consumption in the low- and mid-dose (60 and 175 mg/Kg bw/day) females when compared to the control animals.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in the haematology parameters. Statistically significant differences were considered to be incidental, since there was no relationship with dose and/or all recorded values were near or within the historical control ranges. These differences were not considered to reflect an effect of the test material.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical chemistry findings were observed. All significant differences were considered to be incidental, since there was no relationship with dose and/or all recorded values were near or within the historical control ranges. These differences were not considered to reflect an effect of the test material.

Endocrine findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in the thyroid hormone levels of the male animals. The observed values were in line with the historical control data, no statistically significant or biologically relevant differences were observed between the treated and control groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in the urinalysis parameters.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted during the assessment of grip strength, foot splay or motor activity.

All dose groups of males and females had a normal locomotor activity; the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the treated animals and the control group when evaluating the overall distance travelled (0-60 min, cm). In the high dose male animals, locomotor activity showed an increasing trend between the control/low-dose groups, and through the mid-dose group up until the high-dose group, where the difference reached statistical significance (p<0.05) for the mid- and high-dose groups in the first five minutes (0-5 minutes), and, for the high-dose group, during the 25-30 minutes. For female animals, and for the rest of the male dose groups, the test material did not increase or decrease the normal locomotor activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males:
Treatment-related effects were observed in the liver and kidney weights. The absolute kidney weights were statistically significantly higher compared to control in the mid- and high-dose groups and when adjusted for body weight and brain weights. In high dose males, absolute kidney weights were larger by 22.4% (p<0.01), related to body weights by 24.2% (p<0.01) and by 22.9% (p<0.01) brain related. In mid-dose males, absolute kidney weights were larger by 19.1% (p<0.01). When adjusted to body weights, the kidney weights were higher by 17.6% (p<0.01) and by 18.3% (p<0.01) brain related. The kidney weight changes correlated with histopathological changes in the mid- and high-dose male rats.

Liver weights were statistically significantly higher than control in the mid- (absolute and body weight related) and high-dose (absolute/relative to body and brain) groups. In high-dose males, absolute liver weights were larger by 25.5% (p<0.01), related to body weights by 27.2% (p<0.01) and by 26.0% (p<0.01) brain-related, respectively. In mid-dose males, absolute liver weights were larger by 13.7% (p<0.05), when adjusted to body weights, the liver weights were higher by 12.7% (p<0.01). There was no histopathological correlate for the weight changes in the liver in the high-dose males. No other statistically significant difference was observed among groups in the weights of other organs measured when compared to the control animals.

Females:
Treatment-related effects were observed in the liver and kidney weights. The liver weights relative to body and brain weights were higher (statistically significant) than control in the high-dose groups. Relative to body weights were larger than controls by 27.3% (p<0.01) and 16.9% (p<0.05) brain related. Body weight related liver weights were increased by 9.5% (p<0.05) in the mid-dose group.

The kidney weights relative to body weights were statistically significantly higher than control at the high-dose level. There was an increase of body-related weights by 23.2% (p<0.01). There was no histopathological correlate for the weight changes in kidney and liver in the high-dose females. No other statistically significant difference was observed among groups in the weights of other organs measured when compared to the control animals.

The liver and kidney weight changes were considered to be treatment-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Twelve High dose females were found dead during the study. The macroscopic findings in the females found dead were considered to be agonal or
post-mortem changes.

Necropsy examination did not show any treatment-related change in non-pregnant animals, animals with total intrauterine mortality and their male mating partners. Gross necropsy did not reveal any remarkable findings in the parental animals and all observed changes were considered incidental or a common background.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals found dead:
Histopathological changes were seen in the stomach of two animals found dead. One animal had diffuse minimal submucosal infiltration of inflammatory cells in the non-glandular part of the stomach. One animal had mild focal erosion/ulcer of the non-glandular part of the stomach along with submucosal inflammatory cell infiltration. These findings, which correlated well with the findings from survival animals, were considered to be a treatment-related adverse effect. All other microscopic findings are considered to be post-mortem changes.

Terminal Necropsy (Parental Generation):
Treatment-related findings were observed in the kidney in males and in non-glandular stomach in both sexes.

Minimal to moderate unilateral/bilateral tubular basophilia and minimal to mild accumulation of hyaline droplets were observed in males given 60, 175 or 500 mg/Kg bw/day. Minimal to moderate granular casts were observed in males given 175 or 500 mg/Kg bw/day. The changes in the kidney included hyaline droplet accumulation in the cortical tubules, tubular basophilia and granular casts located at the junction with outer and inner stripes of outer medulla. The hyaline droplets are most likely to be Alpha 2u-globulin which is a low molecular weight protein produced by the liver and excreted through kidney in male rats only (Frazier et al. (2012)). The changes observed in the kidney correlated with the increased kidney weights observed in males given 175 or 500 mg/Kg bw/day. Similar changes were not observed in terminal or found dead high-dose (500 mg/Kg bw/day) females.

Minimal to mild focal erosion/ulcer of the non-glandular stomach was observed in 1/14 high-dose males, 1/3 high-dose females and 1/10 mid-dose females. Minimal to mild squamous cell hyperplasia of the non-glandular stomach was observed in a few treated animals with or without association with erosion/ulcer. Two high-dose females found dead also had lesions in the non-glandular stomach. The single incidence of minimal squamous hyperplasia of the non-glandular stomach in 1/12 low-dose males and 1/10 low-dose females only may be considered within the normal background. The historical control data for this finding in the laboratory (12 studies) did not show any incidence of this finding. However, it has been reported that gastric erosion and ulceration of the glandular or non-glandular stomach is commonly observed in aged rats with more incidence in gavage studies. The edges of the ulcers in the non-glandular stomach often display epithelial hyperplasia (McInnes. E F (2012), Background lesions in laboratory animals, A colour atlas, Saunders, Elsevier, Edinburgh, pp 23.). Occasionally, the non-glandular stomach mucosa of untreated aged rodents exhibit hyperplasia without inflammation or erosion and ulcers. As humans lack a non-glandular stomach, the relevance of the changes produced by chemicals and drugs in the rodent non glandular stomach is often disputed (Greaves, P (2012) Histopathology of preclinical toxicity studies: Interpretation and relevance in drug safety evaluation 4th Edition, Elsevier, Amsterdam pp 343 – 345).

All other changes were seen in control and/or treated animals, or were without meaningful differences in severity and incidence, and therefore regarded as incidental or a common background.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 175 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

2

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 175 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Local Effects

3

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Local Effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

No test material was detected in the control samples. The mean measured test material concentrations of the individual test material containing dose formulations varied between 92.0% and 99.8%. These results were within the acceptable range (90% - 110%) and were considered suitable for the study purposes. All test material formulation samples were found to be homogeneous.

Table 6. Analytical Results
Dose level (mg/kg bw/day)	Nominal concentration (mg/mL)	Measured concentration (mg/mL)	Percentage of nominal concentration (%)
Analytical sampling #1 (Sampling: 18 October 2018)
0	Control	<LOQ	-
60	12	11.78 ± 0.04	98.2
175	35	33.99 ± 0.63	97.1
500	100	92.01 ± 5.90	92.0
Analytical sampling #2 (Sampling:14 November 2018)
0	Control	<LOQ	-
60	12	11.85 ± 0.79	98.7
175	35	34.95 ± 0.91	99.8
500	100	95.67 ± 4.83	95.7
Analytical sampling #3 (Sampling: 05 December 2018)
0	Control	<LOQ	-
60	12	11.52 ± 1.63	96.0
175	35	34.15 ± 0.68	97.6
500	100	98.88 ± 2.76	98.9

Notes: Mean measured concentrations with the 95% confidence intervals are shown.

The LOQ was 3 μg/mL for the analytical samples, which equals to 3.7 mg/mL formula concentration (counting with 1333xdilution during the sample preparation).

N/A: Not applicable

Table 7. Summary of Total Incidence of Clinical Signs (Evaluated Animals)
From Day 0 to Day 61	Male Rats				Females Rats
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Total Number of Animals:	12	12	12	14	10	10	10	3
% of no abnormalities detected:	100.0	100.0	100.0	100.0	60.0	40.0	10.0	0.0
% of Animals with Symptoms:	0.0	0.0	0.0	0.0	40.0	60.0	90.0	100.0
Normal
Number of Animals Affected	12	12	12	14	10	10	10	3
% of Affected Animals	100	100	100	100	100	100	100	100
Number of Times Recorded	348	348	348	407	526	495	511	52
Discharge coloured
Number of Animals Affected	0	0	0	0	0	0	1	0
% of Affected Animals	0	0	0	0	0	0	10	0
Number of Times Recorded	0	0	0	0	0	0	1	0
Faeces liquid
Number of Animals Affected	0	0	0	0	0	2	0	0
% of Affected Animals	0	0	0	0	0	20	0	0
Number of Times Recorded	0	0	0	0	0	38	0	0
Fur thin
Number of Animals Affected	0	0	0	0	0	0	1	0
% of Affected Animals	0	0	0	0	0	0	10	0
Number of Times Recorded	0	0	0	0	0	0	1	0
Hunched Back
Number of Animals Affected	0	0	0	0	0	1	1	3
% of Affected Animals	0	0	0	0	0	10	10	100
Number of Times Recorded	0	0	0	0	0	11	1	39
Noisy respiration
Number of Animals Affected	0	0	0	0	0	0	4	3
% of Affected Animals	0	0	0	0	0	0	40	100
Number of Times Recorded	0	0	0	0	0	0	5	39
Piloerection
Number of Animals Affected	0	0	0	0	4	6	8	3
% of Affected Animals	0	0	0	0	40	60	80	100
Number of Times Recorded	0	0	0	0	8	31	16	92
Red discharge
Number of Animals Affected	0	0	0	0	0	0	0	1
% of Affected Animals	0	0	0	0	0	0	0	33
Number of Times Recorded	0	0	0	0	0	0	0	1

Table 8. Summary of Total Incidence of Clinical Signs (Excluded Animals)
From Day 0 to Day 50	Male Rats				Females Rats
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Total Number of Animals:	-	-	-	-	2	2	2	13
% of no abnormalities detected:	-	-	-	-	100.0	50.0	50.0	0.0
% of Animals with Symptoms:	-	-	-	-	0.0	50.0	50.0	100.0
Normal
Number of Animals Affected	-	-	-	-	2	2	2	13
% of Affected Animals	-	-	-	-	100	100	100	100
Number of Times Recorded	-	-	-	-	94	85	87	46
Activity decreased
Number of Animals Affected	-	-	-	-	0	0	0	2
% of Affected Animals	-	-	-	-	0	0	0	15
Number of Times Recorded	-	-	-	-	0	0	0	6
Ataxia
Number of Animals Affected	-	-	-	-	0	0	0	1
% of Affected Animals	-	-	-	-	0	0	0	8
Number of Times Recorded	-	-	-	-	0	0	0	2
Found Dead
Number of Animals Affected	-	-	-	-	0	0	0	12
% of Affected Animals	-	-	-	-	0	0	0	92
Number of Times Recorded	-	-	-	-	0	0	0	12
Hunched Back
Number of Animals Affected	-	-	-	-	0	0	0	9
% of Affected Animals	-	-	-	-	0	0	0	69
Number of Times Recorded	-	-	-	-	0	0	0	111
Laboured respiration
Number of Animals Affected	-	-	-	-	0	0	0	4
% of Affected Animals	-	-	-	-	0	0	0	31
Number of Times Recorded	-	-	-	-	0	0	0	4
Noisy respiration
Number of Animals Affected	-	-	-	-	0	0	1	11
% of Affected Animals	-	-	-	-	0	0	50	85
Number of Times Recorded	-	-	-	-	0	0	1	123
Piloerection
Number of Animals Affected	-	-	-	-	0	1	0	11
% of Affected Animals	-	-	-	-	0	50	0	85
Number of Times Recorded	-	-	-	-	0	1	0	216
Recumbency
Number of Animals Affected	-	-	-	-	0	0	0	3
% of Affected Animals	-	-	-	-	0	0	0	23
Number of Times Recorded	-	-	-	-	0	0	0	3
Red discharge
Number of Animals Affected	-	-	-	-	0	0	0	5
% of Affected Animals	-	-	-	-	0	0	0	38
Number of Times Recorded	-	-	-	-	0	0	0	11
Tremors Intermittent
Number of Animals Affected	-	-	-	-	0	0	0	1
% of Affected Animals	-	-	-	-	0	0	0	8
Number of Times Recorded	-	-	-	-	0	0	0	2

Table 9. Summary of Bodyweight Data - Male Rats
Day(s) Relative to Start Date		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
0	Mean	476.4 I^1	476.2	472.1	473.9
	SD	31.2	28.9	28.8	37.8
	Max	524	526	524	552
	Min	416	430	424	412
	N	12	12	12	14
	% diff	-	-0.1	-0.9	-0.5
7	Mean	496.4 I^1	499.6	498.0	487.7
	SD	34.7	33.3	32.3	38.3
	Max	545	553	542	560
	Min	438	449	436	424
	N	12	12	12	14
	% diff	-	0.6	0.3	-1.8
14	Mean	517.9 I^1	528.3	524.3	509.1
	SD	33.2	34.5	36.6	42.5
	Max	562	575	578	575
	Min	452	478	463	437
	N	12	12	12	14
	% diff	-	2.0	1.2	-1.7
21	Mean	519.7 I^1	532.8	531.8	521.6
	SD	35.6	32.8	35.8	43.2
	Max	563	578	583	593
	Min	451	492	474	447
	N	12	12	12	14
	% diff	-	2.5	2.3	0.4
27	Mean	535.3 I^1	543.9	545.3	529.8
	SD	35.7	34.9	37.1	40.6
	Max	582	587	598	602
	Min	467	489	481	454
	N	12	12	12	13
	% diff	-	1.6	1.9	-1.0

Statistical Test: Citoxlab DT Transformation: Automatic

[I - Automatic Transformation: Identity (No Transformation)]

Table 10. Summary of Bodyweight Data - Female Rats
		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
From Day 0 to Mating Period (Day(s) Relative to Start Date)
Day 0	Mean	277.7 I^1	281.4	281.6	282.1
	SD	19.7	21.5	17.6	19.6
	Max	308	311	310	317
	Min	245	246	248	242
	N	12	12	12	16
	% diff	-	1.4	1.4	1.6
Day 7	Mean	286.1 I^1	288.4	289.5	266.8
	SD	22.8	24.8	25.8	19.3
	Max	324	331	330	296
	Min	254	249	247	234
	N	12	12	12	10
	% diff	-	0.8	1.2	-6.7
Day 14	Mean	292.1 I^1	293.3	297.3	293.2
	SD	23.5	24.9	24.6	16.8
	Max	331	332	337	311
	Min	260	252	246	261
	N	12	12	12	10
	% diff	-	0.4	1.8	0.4
Gestation Period (Day(s) Relative to Mating (Litter: A)
Day 0	Mean	298.2 I^1	297.0	298.8	288.2
	SD	24.8	25.1	26.6	16.9
	Max	346	341	346	310
	Min	263	252	246	255
	N	12	12	12	10
	% diff	-	-0.4	0.2	-3.3
Day 7	Mean	330.8 I, a^2	335.3	335.3	303.7 d^4
	SD	25.3	28.1	23.3	20.8
	Max	377	381	375	332
	Min	300	296	294	272
	N	12	12	12	10
	% diff	-	1.4	1.3	-8.2
Day 14	Mean	363.2 I, a^2	370.2	363.2	329.7
	SD	34.0	36.6	25.1	28.2
	Max	426	420	403	364
	Min	303	291	320	285
	N	12	12	12	9
	% diff	-	1.9	0.0	-9.2
Day 20	Mean	435.9 R^3	438.8	425.0	382.4
	SD	60.9	64.7	48.4	48.9
	Max	522	526	473	440
	Min	302	296	328	307
	N	12	12	12	9
	% diff	-	0.7	-2.5	-12.3
Lactation Period (Day(s) Relative to Littering (Litter: A))
Day 0	Mean	355.8 I^1	364.4	346.2	319.7
	SD	29.8	25.3	26.7	22.6
	Max	408	402	390	341
	Min	311	328	299	296
	N	10	10	9	3
	% diff	-	2.4	-2.7	-10.2
Day 4	Mean	368.3 I^1	378.4	353.6	342.3
	SD	29.7	31.8	26.7	37.9
	Max	422	416	390	369
	Min	320	326	304	299
	N	10	10	9	3
	% diff	-	2.7	-4.0	-7.1
Day 13	Mean	406.6 I^1	397.9	388.4	369.3
	SD	28.9	42.2	29.4	29.0
	Max	460	445	411	398
	Min	357	326	315	340
	N	10	10	9	3
	% diff	-	-2.1	-4.5	-9.2

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

3 [R - Automatic Transformation: Rank]

4 [d - Test: Dunnett 2 Sided p < 0.05]

Table 11. Summary of Bodyweight Gain Data - Male Rats
Day(s) Relative to Start Date		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
0 to 7	Mean	20.0^1	23.4	25.9	13.8
	SD	8.7	6.3	8.0	6.9
	Max	37	35	40	27
	Min	4	15	12	-2
	N	12	12	12	14
	% diff	-	17.1	29.6	-31.1
7 to 14	Mean	21.5 I^2	28.8	26.3	21.4
	SD	12.0	6.3	7.6	10.5
	Max	36	37	38	39
	Min	1	15	14	2
	N	12	12	12	14
	% diff	-	33.7	22.5	-0.7
14 to 21	Mean	1.8^1	4.4	7.5	12.6 dd^3
	SD	6.3	5.5	8.4	7.6
	Max	11	15	24	28
	Min	-11	-6	-3	-5
	N	12	12	12	14
	% diff	-	152.4	328.6	618.4
21 to 27	Mean	15.6 I^2	11.2	13.4	13.7
	SD	7.6	7.8	6.0	7.6
	Max	24	22	23	28
	Min	-4	-7	5	5
	N	12	12	12	13
	% diff	-	-28.3	-13.9	-12.1
0 to 27	Mean	58.8 I^2	67.8	73.2	61.9
	SD	14.0	12.8	14.9	19.8
	Max	71	90	98	96
	Min	20	46	47	29
	N	12	12	12	13
	% diff	-	15.2	24.4	5.3

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

2 [I - Automatic Transformation: Identity (No Transformation)]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 12. Summary of Bodyweight Gain Data - Female Rats: From Day 0 to Mating Period
Day(s) Relative to Start Date		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Day 0 to 7	Mean	8.4^1	7.0	7.9	-15.0 dd^3
	SD	7.8	7.5	13.1	14.2
	Max	20	20	34	7
	Min	-4	-5	-10	-40
	N	12	12	12	10
	% diff	-	-16.8	-5.9	-278.2
Day 7 to 14	Mean	6.0^1	4.9	7.8	26.4 dd^3
	SD	7.0	11.7	9.4	11.8
	Max	15	25	21	46
	Min	-8	-15	-9	6
	N	12	12	12	10
	% diff	-	-18.1	30.6	340.0
Day 0 to 14	Mean	14.4 I^2	11.9	15.8	11.4
	SD	8.6	10.1	11.5	13.3
	Max	29	25	32	30
	Min	-2	-9	-4	-8
	N	12	12	12	10
	% diff	-	-17.3	9.2	-20.9

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

2 [I - Automatic Transformation: Identity (No Transformation)]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 13. Summary of Bodyweight Gain Data - Female Rats: Gestation Period
Day(s) Relative to Mating (Litter: A)		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Day 0 to 7	Mean	32.7^1	38.3	36.5	15.5
	SD	8.5	13.1	13.5	15.1
	Max	43	50	54	31
	Min	13	6	1	-20
	N	12	12	12	10
	% diff	-	17.3	11.7	-52.6
Day 7 to 14	Mean	32.3 R, k^2	34.8	27.9	25.3
	SD	16.4	13.6	13.0	7.2
	Max	57	47	43	34
	Min	-4	-5	-5	13
	N	12	12	12	9
	% diff	-	7.7	-13.7	-21.6
Day 14 to 20	Mean	72.8 R^3	68.7	61.8	52.8
	SD	33.0	37.0	37.2	25.4
	Max	96	110	93	76
	Min	-1	-12	2	1
	N	12	12	12	9
	% diff	-	-5.6	-15.0	-27.5
Day 0 to 20	Mean	137.8 R, k^2	141.8	126.3	94.7 u^4
	SD	50.2	59.1	53.1	44.7
	Max	177	207	172	139
	Min	30	6	17	-3
	N	12	12	12	9
	% diff	-	3.0	-8.3	-31.3

Statistical Test: Citoxlab DT Transformation: Automatic

1 [R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.01]

2 [R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05]

3 [R - Automatic Transformation: Rank]

4 [u - Test: Dunn 2 Sided p < 0.05]

 

Table 14. Summary of Bodyweight Gain Data - Female Rats: Lactation Period
(Day(s) Relative to Littering (Litter: A))		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Day 0 to 4	Mean	12.5 I^1	14.0	7.3	22.7
	SD	8.0	17.1	5.9	17.6
	Max	22	42	16	37
	Min	-6	-13	-3	3
	N	10	10	9	3
	% diff	-	12.0	-41.3	81.3
Day 4 to 13	Mean	38.3 R^2	19.5	34.9	27.0
	SD	9.2	25.0	16.0	15.1
	Max	51	61	58	41
	Min	24	-36	11	11
	N	10	10	9	3
	% diff	-	-49.1	-8.9	-29.5
Day 0 to 13	Mean	50.8 R^2	33.5	42.2	49.7
	SD	12.9	30.7	16.1	6.7
	Max	73	70	62	57
	Min	32	-41	16	44
	N	10	10	9	3
	% diff	-	-34.1	-16.9	-2.2

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [R - Automatic Transformation: Rank]

Table 15. Food Mean Consumption (g/animal/day): Male Rats
		Day(s) Relative to Start Date
		0 to 7	7 to 14	14 to 21	21 to 27
Group 1 0 mg/Kg bw/day	Mean	27.73 I, a^1	27.21 R^2	25.17 R^2	25.64 I^3
Group 2 60 mg/Kg bw/day	Mean	27.26	26.98	25.76	26.32
Group 3 175 mg/Kg bw/day	Mean	27.02	27.04	27.40	28.07
Group 4 500 mg/Kg bw/day	Mean	25.20 dd^4	26.26	26.69	27.64

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

2 [R - Automatic Transformation: Rank]

3 [I - Automatic Transformation: Identity (No Transformation)]

4 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 16. Food Mean Consumption (g/animal/day): Female Rats: From Day 0 to Mating Period
		Day(s) Relative to Start Date
		0 to 7	7 to 14
Group 1 0 mg/Kg bw/day	Mean	18.27^1	17.79^1
Group 2 60 mg/Kg bw/day	Mean	19.12	18.90
Group 3 175 mg/Kg bw/day	Mean	18.29	20.04 dd^3
Group 4 500 mg/Kg bw/day	Mean	9.63 dd^3	17.63

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 17. Food Mean Consumption (g/animal/day): Female Rats: Gestation Period
		Day(s) Relative to Start Date
		0 to 7	7 to 14	14 to 21
Group 1 0 mg/Kg bw/day	Mean	23.96^1	24.94^1	27.33 I, a^2
Group 2 60 mg/Kg bw/day	Mean	25.81	27.77	28.85
Group 3 175 mg/Kg bw/day	Mean	25.14	25.77	27.28
Group 4 500 mg/Kg bw/day	Mean	18.66 dd^3	20.02 dd^3	22.24

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

2 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 18. Food Mean Consumption (g/animal/day): Female Rats: Lactation Period
		Day(s) Relative to Start Date
		0 to 7	7 to 13
Group 1 0 mg/Kg bw/day	Mean	42.49 R^1	69.37 R^1
Group 2 60 mg/Kg bw/day	Mean	45.56	74.03
Group 3 175 mg/Kg bw/day	Mean	40.60	67.19
Group 4 500 mg/Kg bw/day	Mean	39.90	57.72

Statistical Test: Citoxlab DT Transformation: Automatic

1 [R - Automatic Transformation: Rank]

Table 19. Select Haematology Parameters
Haematology Parameter	Groups/Concentration (mg/kg bw/day)
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Males
APTT (sec)	11.26	11.44	10.32	11.82
	Differences from control	1.6%	-8.3%	5.0%
	Historical control data (mean ± 2SD)	12.33 ± 1.260
PTT (sec)	10.40	10.26	10.00	10.14
	Differences from control	-1.3%	-3.8%	-2.5%
	Historical control data (mean ± 2SD)	9.86 ± 0.324
Females
APTT (sec)	13.08	12.66	11.98	11.23*
	Differences from control	-3.2%	-8.4%	-14.1%
	Historical control data (mean ± 2SD)	12.95 ± 2.017
PTT (sec)	9.26	8.92	8.70++	8.80
	Differences from control	-3.7%	-6.0%	-5.0%
	Historical control data (mean ± 2SD)	8.86 ± 0.489

*= p<0.05, **= p<0.01; Dunnett two-sided test.

+= p<0.05, ++= p<0.01; Dunn two-sided test.

 

Table 20. Select Clinical Chemistry Parameters
Clinical Chemistry Parameter	Groups/Concentration (mg/kg bw/day)
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Males
Albumin (g/L)	30.98	30.88	32.14	33.70+
	Differences from control	-0.3%	3.7%	8.8%
	Historical control data (mean ± 2SD)	30.02 ± 2.053
A/G Ratio	1.36	1.42	1.44	1.56**
	Differences from control	4.4%	5.9%	14.7%
	Historical control data (mean ± 2SD)	1.21 ± 0.093
Alkaline Phosphatase (U/L)	82.8	86.6	84.0	88.6
	Differences from control	4.6%	1.4%	7.0%
	Historical control data (mean ± 2SD)	99.7 ± 24.99
Females
Albumin (g/L)	31.20	31.56	34.14	32.57
	Differences from control	1.2%	9.4%	4.4%
	Historical control data (mean ± 2SD)	29.54 ± 2.126
A/G Ratio	1.25	1.30	1.42	1.30
	Differences from control	4.0%	13.6%	4.0%
	Historical control data (mean ± 2SD)	1.16 ± 0.086
Alkaline Phosphatase (U/L)	72.3	121.6**	87.4	95.0
	Differences from control	68.3%	21.0%	31.5%
	Historical control data (mean ± 2SD)	88.4 ± 27.22

*= p<0.05, **= p<0.01; Dunnett two-sided test

+= p<0.05, ++= p<0.01; Dunn two-sided test

 

Table 21. Kidney and Liver Organ Weights
Organ weights	Groups/Concentration (mg/kg bw/day)
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Males
Terminal bodyweight (g)	508.8	513.7	516.0	503.1
	Differences from control	1.0%	1.4%	-1.1%
	Historical control data (mean ± 2SD)	478.5 ± 35.75
Liver (g)	15.640	15.984	17.788*	19.623**
	Differences from control	2.2%	13.7%	25.5%
	Historical control data (mean ± 2SD)	13.272 ± 1.4813
Liver / Bodyweight (%)	3.059	3.107	3.448**	3.892**
	Differences from control	1.6%	12.7%	27.2%
	Historical control data (mean ± 2SD)	2.7721 ± 0.21044
Liver / Brain weight (%)	692.22	707.13	783.36	872.10**
	Differences from control	2.2%	13.2%	26.0%
	Historical control data (mean ± 2SD)	602.6183 ± 70.57230
Kidney (g)	3.346	3.523	3.984**	4.096**
	Differences from control	5.3%	19.1%	22.4%
	Historical control data (mean ± 2SD)	3.065 ± 0.2627
Kidney / Bodyweight (%)	0.657	0.686	0.773**	0.816**
	Differences from control	4.4%	17.6%	24.2%
	Historical control data (mean ± 2SD)	0.6417 ± 0.04463
Kidney / Brain weight (%)	148.19	155.69	175.29**	182.07**
	Differences from control	5.1%	18.3%	22.9%
	Historical control data (mean ± 2SD)	139.1606 ± 12.57275
Females
Terminal bodyweight (g)	363.8	363.0	352.1	314.7
	Differences from control	-0.2%	-3.2%	-13.5%
	Historical control data (mean ± 2SD)	355.5 ± 23.52
Liver (g)	13.660	14.156	14.443	15.077
	Differences from control	3.6%	5.7%	10.4%
	Historical control data (mean ± 2SD)	13.698 ± 1.2264
Liver / Bodyweight (%)	3.762	3.903	4.119*	4.788**
	Differences from control	3.7%	9.5%	27.3%
	Historical control data (mean ± 2SD)	3.8577 ± 0.29215
Liver / Brain weight (%)	656.43	681.44	719.72	767.17*
	Differences from control	3.8%	9.6%	16.9%
	Historical control data (mean ± 2SD)	684.7389 ± 62.85047
Kidney (g)	2.234	2.367	2.375	2.387
	Differences from control	6.0%	6.3%	6.8%
	Historical control data (mean ± 2SD)	2.209 ± 0.1924
Kidney / Bodyweight (%)	0.616	0.656	0.676	0.759**
	Differences from control	6.5%	9.6%	23.2%
	Historical control data (mean ± 2SD)	0.6217 ± 0.03948
Kidney / Brain weight (%)	107.46	113.98	118.25	121.74
	Differences from control	6.1%	10.0%	13.3%
	Historical control data (mean ± 2SD)	110.4571 ± 10.25619

*= p<0.05; **= p<0.01; Dunnett two-sided test.

Table 22. Incidence of Treatment-related Microscopic Changes in the Kidney
	Males				Females
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Number of animals	12	12	12	14	10	10	10	3
Accumulation, Hyaline droplets, bilateral/unilateral
Minimal	0	1	4	3
Mild	0	0	5	9	0	0	0	0
Total	0	1	9	12	0	0	0	0
Basophilia, bilateral/unilateral
Minimal	0	5	3	2	0	0	0	0
Mild	0	4	6	7	0	0	0	0
Moderate	0	0	3	5	0	0	0	0
Total	0	9	12	14	0	0	0	0
Cast, granular, bilateral /unilateral
Minimal	0	0	1	2	0	0	0	0
Mild	0	0	3	5	0	0	0	0
Moderate	0	0	2	1	0	0	0	0
Total	0	0	6	8	0	0	0	0
Number of tissues examined	12	12	12	14	5	0	0	3

*Note: Data of two non-pregnant females in the Control Goup, two females with total intrauterine mortality in the Low dose group, two females with total intrauterine mortality in the Mid dose group, and 12 found dead females and one female with total intrauterine mortality in the High dose group were excluded from this evaluation.

Table 23. Incidence of Treatment-related Microscopic Changes in the Stomach
	Males				Females
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Number of animals	12	12	12	14	10	10	10	3
Erosion/ulcer, non-glandular region
Minimal	0	0	0	0	0	0	1	0
Mild	0	0	0	1	0	0	0	1
Total	0	0	0	1	0	0	1	1
Hyperplasia, Squamous, non-glandular region
Minimal	0	1	5	5	0	1	3	0
Mild	0	0	0	1	0	0	0	1
Total	0	1	5	6	0	1	3	1
Infiltrate, Inflammatory Cells, non-glandular region
Minimal	0	0	1	1	0	0	1	0
Mild	0	0	0	1	0	0	0	1
Total	0	0	1	2	0	0	1	1
Number of tissues examined	12	12	12	14	10	10	10	3

*Note: Data of two non-pregnant females in the Control Goup, two females with total intrauterine mortality in the Low dose group, two females with total intrauterine mortality in the Mid dose group, and 12 found dead females and one female with total intrauterine mortality in the High dose group were excluded from this evaluation.

Table 24. Summary of Thyroid Hormone Concentrations (Parental Males)
	Day(s) Relative to Start Date		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Adult T4 Conc. (ng/mL)	203 to 208	Mean	42.89 R^+	42.43	41.95	41.52
		SD	3.76	4.67	4.92	7.35
		Max	50.8	50.1	50.0	54.8
		Min	35.0	35.3	33.7	31.5
		N	12	12	12	14
		% Diff	-	-1.1	-2.2	-3.2

Applicant's summary and conclusion

Conclusions:

Based on the effects observed, the systemic No Observed Adverse Effect Level (NOAEL) for Ocimene PQ [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene] was determined to be 175 mg/Kg bw/day, based on mortality observed in female rats at the highest dose tested. The NOAEL for local toxicity (stomach effects) was determined to be 60 mg/Kg bw/day and 175 mg/Kg bw/day for female and male rats, respectively.

Executive summary:

In a key combined repeated dose and reproductive/developmental toxicity screening study, the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene; EC# 933-779-9) was administered via daily oral gavage to male and female Wistar rats at doses of 0, 60, 175, or 500 mg/Kg bw/day (in propylene glycol with 1% polysorbate 80 vehicle). Rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males while the females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13.

 

Signs of morbidity and mortality were observed for twice daily while general and detailed observations for clinical signs were undertaken daily and weekly, respectively. Body weight and food consumption were measured weekly and clinical pathology evaluations, including haematology, coagulation, clinical chemistry and urinalysis were undertaken. Neurological assessments including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. At termination, necropsy with macroscopic examination was performed. Weights of selected organs recorded, and representative tissues/organs sampled and preserved in appropriate fixatives from the adult animals. Thyroxine (T4) levels in adult males were also assessed. For adult animals, a detailed histological examination was performed on a select list of retained organs in the control and high dose groups and for all those male / female mating pairs where no conceptus and/or no live born pups was achieved. Track down examination of stomach samples of all remaining animals and kidneys of all remaining male rats was also performed.

 

Treatment-related mortality was observed in the high-dose females (12 out of 16 animals), either at the beginning of the treatment (between Days 2-6) or near the end of the pregnancies (on GD20-22). No further mortality, clinically adverse effects (except for minor, transient symptoms), or changes in neurological assessment were observed through the study period. Adverse effects on the body weight of females in the high dose (500 mg/Kg bw/day) group were observed during the gestation (reaching statistical significance) and lactation period, with similar changes observed in food consumption.

 

Haematology, clinical chemistry, and urinalysis parameters evaluated remained unaffected by treatment and no treatment-related changes were observed in the thyroid hormone levels of the adult male animals. Gross necropsy did not reveal any remarkable treatment-related adverse effects.

 

Higher liver and kidney weights were observed in both sexes in the mid- and high-dose animals (175 and 500 mg/Kg bw/day). These organ weight changes were correlated with the microscopic kidney findings in the males (most likely to be Alpha 2u globulin which is specific to male rat kidney only). These changes were considered to be non-adverse in the context of human health. Histopathological findings in the stomach included mild focal erosion/ulcer of the nonglandular stomach in 1 out of 14 high-dose males and in 1 out of 3 high-dose females; minimal focal erosion/ulcer in the same region in 1 out of 10 mid-dose females. These observations were considered to be an adverse, treatment-related local irritant effect.

 

Based on the effects observed, the systemic No Observed Adverse Effect Level (NOAEL) for Ocimene PQ [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene] was determined to be 175 mg/Kg bw/day, based on mortality observed in female rats at the highest dose tested. The NOAEL for local toxicity (stomach effects) was determined to be 60 mg/Kg bw/day and 175 mg/Kg bw/day for female and male rats, respectively.